Table of Content 1. Description...2 2. Features...2...2...2 3. Kit components...3 4. Reagents and instruments not supplied in this kit...3 5. Storage...4 6. Principle...4 7. Preparation of RNA sample...5 8. Notes...5 9. Protocol 9-1. General One Step RT-PCR...6 9-2. Two Step RT-PCR (example)...7 10. References...8
1. Description mrna Selective PCR Kit Ver.1.1 For amplification of cdna alone which derives from mrna with simple procedure PCR (Polymerase Chain Reaction) process is a simple and powerful method which allows in vitro amplification of DNA fragments through a succession of three incubation steps at different temperatures. In principle, PCR is a method to amplify DNA segments, and not directly amplify RNA. However, synthesis of cdna from RNA using reverse transcriptase enables to apply PCR process to the RNA analysis, which is RT-PCR. Many reports of various fields have been made by applying RT-PCR, such as of structural analysis of RNA, efficient cdna cloning, analysis of gene expression at the RNA level, etc. The purity of RNA sample will affect the yield of cdna synthesis. Generally AGPC (Acid-Guanidinium-Thiocyanate-Phenol Chloroform) method is widely used for purification of RNA. However, this method cannot completely remove genomic DNA contaminated in sample RNA. When sample RNA is contaminated with genomic DNA, normal RT-PCR has risk that genomic DNA or pseudo gene can be amplified together with cdna of interest. To prevent amplification of genomic DNA or pseudo gene, it is required to treat mrna with DNase, or to purify mrna through a proper method. However, these treatments are laborious and might damage RNA. Use of TaKaRa mrna Selective PCR Kit eliminates these pretreatments, and achieves amplification of only cdna which derives from RNA by incorporation of nucleotide analog in RT and/or PCR reaction and by utilizing specific mrna Selective PCR Buffer. (Refer to the 5. Principle for the details.) Also this kit is designed to perform RT-PCR in "one-step", which enables RT-PCR reaction all in a single tube, utilizing AMV (Avian Myeloblastosis Virus) Reverse Transcriptase and subsequent amplification using AMV-Optimized Taq (which was developed in LA (Long and Accurate) PCR technology*.). This kit can be available for two-step RT-PCR as well, with use of Oligo dt Primer and Random 9 mers. All reagents necessary for the reverse transcription and subsequent PCR being included, this kit is useful for simple and efficient analysis of RNA. * U.S. Patent 5,436, 149 for LA Technology is owned by 2. Features Available RNA as template RNA segment to be transcribed and later amplified Reverse Transcriptase DNA Polymerase RNase Inhibitor General 2 kbp AMV Reverse Transcriptase (in the range of 42-50 ) AMV-Optimized Taq Supplied in the kit 2 Protocol Single tube reaction in one-step (*Also available for two-step method)
3. Kit components (50 reactions) 1. AMV Reverse Transcriptase XL *1 (5 units/ μ l) 50 μ l (originated from Avian Myeloblastosis Virus) 2. RNase Inhibitor (40 units/ μ l) 50 μ l 3. AMV-Optimized Taq (5 units/ μ l) 50 μ l 4. 2 x mrna Selective PCR Buffer I *2 1.25 ml 5. 2 x mrna Selective PCR Buffer II *2 1.25 ml 6. MgCl 2 (25 mm) 500 μ l 7. dntp/analog mixture *3 250 μ l 8. Control F-1 primer *4 (20 μ M) 10 μ l 9. Control R-1 primer *4 (20 μ M) 10 μ l 10. Control Template *5 10 μ l 11. RNase Free distilled H 2 O 1 ml 12. Random 9 mers *6 (50 μ M) 50 μ l 13. Oligo dt primer *6 (50 μ M) 50 μ l *1 Manufactured by Life Sciences, Inc. *2 For general reactions, 2 x mrna Selective PCR Buffer is used. In case that the GC content of template RNA is low (<40%) and amplification is difficult, it is advisable to use 2 x mrna Selective PCR Buffer II. *3 dntp/analog mixture is prepared by addition of dntp analog into dntp mixture which contains four dntps at each 10 mm. *4 Primers Sequence Control F-1 primer: 5'-CGTCTAACAGTGGAAGCTGATC-3' Control R-1 primer: 5'-CATGACTTCATACTTCTGCCTC-3' *5 Control Template Supplied Control template contains Total RNA derived from cultured human HT29 cells and genomic DNA. Only the amplified fragment of 252 bp which derives from RNA is obtained by following the protocol of this kit. When this template is reverse-transcribed and is amplified with Takara's One Step RNA PCR Kit (#RR024), two amplified fragments are obtained; one is 252 bp derived from RNA, and the other is 604 bp from genomic DNA. *6 Oligo dt Primer, Random 9 mers These two primers are used in two-step method, not in one-step method. 4. Reagents and instruments not supplied in this kit 1. Mineral Oil 2. Agarose gel ex. NuSieve ィ 3:1 Agarose (Cambrex Cat#50091, 50092, 50094) 3. Upstream primer and downstream primer 4. Authorized thermal cycler for PCR 5. Microcentrifuge tubes (made of polypropylene) 6. Agarose gel electrophoresis apparatus 7. Microcentrifuge 8. Micropipets and pipette tips (autoclaved) 3
5. Storage - 20 6. Principle when nucleotide analog is incorporated during RT and/or PCR reaction, Tm declines and denaturation at low temperature (85 C) becomes possible. In this case, PCR reaction can proceed with low denaturation temperature and only cdna with low Tm value is amplified. Whereas genomic DNA can not be denatured at low temperature, so it can not be used as PCR template or can not be amplified. mrna Genomic DNA * Nucleotide analog Reverse transcription with nucleotide analog substrate (Nucleotide analog is incorporated.) Denaturation at 85 As genomic DNA not containing nucleotide analog cannot be denatured at 85, it cannot work as PCR templates. Annealing PCR reaction with a speciffic buffer Extension Denaturation at 85 Amplified products X PCR cannot be achieved. PCR can be achieved. Fig. 1 Principle of mrna Selective PCR Kit 4
7. Preparation of RNA sample TaKaRa mrna Selective PCR Kit (AMV) is designed to perform the reverse transcription of RNA to cdna and subsequent amplification. The purity of RNA sample will affect the yield of cdna synthesis. So it is essential to inhibit the activity of RNase in the cells and also to prevent the contamination of RNase derived from equipments and solutions used. Extra precautions should be taken during the sample preparation; put on clean disposable gloves, dedicate a table to exclusive use for RNA preparation, and avoid unnecessary talks during the operations to prevent the contamination of RNase from operators' sweat or saliva. A. Equipment Disposable plastic equipments should be used. In case using glass tools, treat the glass tools with DEPC (diethylpyrocarbonate) prior to use. (1) Treat glass tools with 0.1% DEPC solution at 37, 12 hours. (2) Autoclave at 120, 30 min., to remove DEPC left on the tools. It is recommended to prepare all the equipments as the exclusive use for RNA preparation. B. Reagent Reagents for RNA preparation, including distilled water, should be prepared with heat sterilized glass tools (180, 60 min.), or if possible those treated with 0.1% DEPC solution and autoclaved. Reagents and distilled water should be exclusively used for RNA preparation. C. Preparation method Simple purification methods can yield enough amount of RNA for reverse transcription and subsequent PCR. However, it is recommended to use highly purified RNA obtained by AGPC (Acid-Guani dine-thiocyanate- Phenol-Chloroform) method, etc. 8. Note (1) For both reverse transcription and PCR amplification, master mix of reagents (containing RNase-free sterilized distilled water, buffers, dntp mixtures, MgCl 2 solution, etc) for all samples can be prepared first, then aliquoted to individual tubes. Using such mixtures will allow accurate reagents dispense: minimize reagents pipetting losses, and avoid repeat dispensing of hte each reagent. This helps to minimize variation of the data among the experiments. (2) Enzymes such as RTase, AMV-Optimized Taq, and RNase Inhibitor shall be mixed gently by pipetting. Avoid generating bubbles. Gently spin down the solution prior to mixing. Pipette enzymes carefully and slowly as the viscosity of the 50% glycerol in the buffer can lead to pipetting errors. (3) Keep enzymes at - 20 until ust just before use and return into the freezer promptly after use. 5
(4) Please avoid freeze-thawing cycle for the positive control RNA. It is recommended to store the positive control RNA in aliquots at - 70 ~ - 80. (5) Use new disposable pipette tips to avoid contamination between samples. 9. Protocol 9-1. General One-Step RT-PCR 1. Prepare the reaction mixture in a tube by combining the reagents in the proportions shown. Reagents Volume Final concentration [or Volume to be added] 2 x mrna Selective PCR Buffer I or II 25 μ l x 1x 25 mm MgCl 2 10 0 μ l 5 mm dntp/analog mixture 5 μ l 1 mm RNase Inhibitor (40 units/ μ l) 1 μ l 0.8 units/ μ l AMV RTase XL (5 units/ μ l) 1 μ l 0.1 0. unit/ μ l AMV-Optimized Taq (5 units/ μ l) 1 μ l 0.1 0. unit/ μ l Specific upstream primer (20 μ M) 1 μ l 0.4 μ M (sense primer) Specific downstream primer (20 μ M) 1 μ l 0.4 μ M (anti-sense primer) Control Template or 1 μ l or * Experimental Sample 1 μ g of total RNA RNase Free distilled H 2 O 4 μ l Total volume 50 μ l * In case that the expression level of RNA is low, the volume to be added can be increased up to 5 μ l. 2. Overlay mineral oil* (50~100 μ l) to avoid the evaporation of the reaction mixture. * Some thermal cyclers do not require mineral oil. 3. Place all tubes in a Thermal Cycler and set the parameters by the following condition. General condition 42-50 15-30 min. Reverse 50 15 min. transcription 85 * 0.5-1 min. 85 1 min. 37-65 0.5-1 min. PCR 45 1 min. In the case of control experiment using Control Template 25-30 cycles 25 cycles 72 1-5 min. 72 1 min. 6
* When the reaction does not proceed, raise or lower the denaturation temperature by 1 C intervals in the range of 80-90. ** Set the aneal temperature 5-10 lower than general condition. Note: 1) When more amount of genomic DNA is contaminated in sample RNA, amplified fragments derived from genomic DNA may appear. Even using this kit, it is recommended to prepare highly-purified RNA sample with AGPC method. 2) Too many cycles may generate amplified fragments derived from genomic DNA. 3) DNase treatment can cause fragmentation of genomic DNA, which leads to amplification of genomic DNA. Therefore, it is not recommended to treat a sample with DNase. 4) PCR Condition The extension time depends on the length of the target cdna. Usually AMV-Optimized Taq extends DNA at the 500 bases per minute. 4. After the amplification is completed, apply 5-10 μ l of the reactant for agarose gel electrophoresis to verify the amplified DNA fragments.* * The PCR amplified product samples can be stored frozen until subsequent analysis. In the control experiment target cdna can be verified by the amplified fragment of 252 bp. 9-2. Two-Step RT-PCR reaction (example) Prepare the reaction mixtures in a tube by combining the reagents in the proportions as shown, and perform reactions. [RT reaction] Composition of reaction mixture Reagents Volume Final concentration [or Volume to be added] 2 x mrna Selective PCR Buffer I or II 25 μ l 1x 25 mm MgCl 2 0 10 μ l 5 mm dntp/analog mixture 5 μ l 1 mm RNase Inhibitor (40 units/ μ l) 1 μ l 0.8 units/ μ l AMV RTase XL (5 units/ μ l) 1 μ l 0.1 unit/ μ l Oligo dt primer, or Random 9 mers, 1 μ l or Specific primer (20 μ M) Control Template or 1 μ l or * Experimental Sample 1 μ g of total RNA RNase Free distilled H 2 O 6 μ l Total volume 50 μ l * In case that the expression level of RNA is low, the volume to be added can be increased up to 7 μ l. Reaction condition 30 10 min. 42-50 15-30 min. 1 Cycle 5 5 min. 7
[PCR reaction] Composition of reaction mixture Reagents Volume Final concentration [including the amount of RT reactant] 2 x mrna Selective PCR Buffer I or II 20 μ l 1x 25 mm MgCl 2 8 μ l 5 mm dntp/analog mixture 4 μ l 1 mm AMV-Optimized Taq (5 units/ μ l) 1 μ l 0.1 unit/ μ l Upstream specific primer (20 μ M) 1 μ l 0.4 μ M (sense primer) Downstream specific primer (20 μ M) 1 μ l 0.4 μ M (antisense primer) RT reactant 10 0 μ l Sterilized distilled H 2 O 5 μ l Total volume 50 μ l Reaction condition 85 0.5-1 min. 37-65 0.5-1 min. 25-30 cycles 72 1-5 min. 11. Reference 1) Kawasaki, E.S. and Wang, A.M. (1989) PCR Technology (Erilch, H.A.ed) Stockton Press 89-97. 2) Lynas, C., Cook, S.D., Laycock, K.A., Bradfield, J.W.B., and Maitland, N.J. (1989) J. Pathnology. 157, 285-289. 3) frohman, M.A., Dush, M.K., Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002. 4) Mallet, M., Oriol, G., Mary, C., Verrier, B. and Mandrand, B. (1995) BioTechniques 18 No. 4, 678-687. 5) Tatiana, A., John J. Snimsky., David H. Gelfand and Thomas W. Myers (1996) Nucleic Acids Research 24 (24), 5021-5025. NOTICE TO PURCHASER: LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser's own internal research. No right under any other patent claim (such as the patented 5' Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. U.S. Patent 5,436,149 for LA Technology owned by Use of mrna Selective PCR Kit (AMV) Ver.1.1 is licensed from biomerieux, is covered by US Patent 5,817,465 and equivalents, and is for Research Use Only. NOTE: This product is intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc.takara products may not be resold or transfered, modified for resale or transfer, or used to manufacture commercial products without written approval from If you require licenses for other use, please call at +81 77 543 7247 or contact from our website at www.takara-bio.com 8 Phone: +81-77-543-7247 Fax: +81-77-543-9254